Differently from TTV, ReDoV was not detected in any blood samples. Conclusions: This study establishes the prevalence and mean value of TTV and ReDoV in saliva samples and demonstrates the existence of differences between these two components of the human virome.Objectives: Torquetenovirus (TTV) and Redondovirus (ReDoV) are the most prevalent viruses found in the human respiratory virome in viral metagenomics studies. A large-scale epidemiological study was performed to investigate their prevalence and loads in saliva samples according to SARS-CoV-2 status. Methods: Saliva samples from 448 individuals (73% SARS-CoV-2 negative and 27% SARS-CoV-2 positive) aged 23–88 years were tested. SARS-CoV-2 and TTV were determined in saliva by specific qualitative and quantitative real-time PCRs, respectively. A sub-cohort of 377 subjects was additionally tested for the presence and load of ReDoV in saliva, and a different sub-cohort of 120 subjects for which paired saliva and plasma samples were available was tested for TTV and ReDoV viremia at the same timepoints as saliva. Results: TTV in saliva was 72% prevalent in the entire cohort, at a mean DNA load of 4.6 log copies/mL, with no difference regardless of SARS-CoV-2 status. ReDoV was found in saliva from 61% of the entire cohort and was more prevalent in the SARS-CoV-2-negative subgroup . In saliva, the total mean load of ReDoV was very similar to the one of TTV, with a value of 4.4 log copies/mL. The mean viral loads in subjects infected with a single virus, namely, those infected with TTV or ReDoV alone, was lower than in dually infected samples, and Tukey’s multiple-comparison test showed that ReDoV single-infected samples resulted in the only true outlier ( Torquetenovirus (TTV) and Redondovirus (ReDoV) are viruses with a single, circular DNA (ssDNA) genome approximately 3000 nucleotides (nt) in length and are the most prevalent viruses found in the human respiratory virome in viral metagenomics studies . Both viHowever, despite these similarities, TTV and ReDoV differ widely from each other in genomic organization and genetic variability and represent the prototypes of two distinct viral families: Anelloviridae and Redondoviridae . TTV is ReDoV was discovered in 2018 by metagenomic sequencing and then more widely characterized by specific-PCR investigations . DiffereWhile SARS-CoV-2-positive patients coinfected with common respiratory viruses have been widely described and the effect of a more severe disease among these patients has been established ,12, the Here, 448 saliva samples from SARS-CoV-2-negative and -positive subjects were investigated for TTV and ReDoV DNA presence and loads. The study aimed to investigate if the measurements of these viruses differed in saliva samples, if they varied in presence of SARS-CoV-2 RNA and if the measure of TTV in saliva could mirror the size of the virus circulating in the blood.Saliva samples from 448 individuals  were collected. Of these, 326 (73%) samples were from healthy individuals enrolled during the screening for SARS-CoV-2 infection, and 122 (27%) from patients admitted to the hospital after the diagnosis of COVID-19 provided by real-time RT-PCR on nasopharyngeal swabs . In both cohorts, the subjects were requested to provide saliva through the drooling technique. This technique allows for collecting only oral fluids, thus excluding mucous secretions from the oropharynx or lower respiratory tract . Saliva samples were self-collected in a sterilized 15 mL polystyrene sputum collection tube and transported to the laboratory within 2 h, where they were conserved at −80 °C for a maximum period of 6 months before use. All the 448 saliva samples were tested for SARS-CoV-2 RNA and TTV DNA presence and load. A statistically representative sub-cohort of saliva samples from 377 of 448 subjects  was randomly selected and investigated for ReDoV DNA detection and quantitation. The study was run after ethical approval of ASST Sette Laghi Institutional Review Board (protocol number 68/2020).® Kit  which amplifies the viral RdRP, and ORF8 genes as well as the human RNase P gene as an internal control. Following the manufacturer’s instructions, a master mix was prepared to contain all components of the reaction including enzymes, nucleotides, probes, and fluorophores; 20 microliters of this mix were added per well in a 96-well plate, and 10 microliters of extracted RNA were then added. The PCR program was carried out for 45 cycles at 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 20 s. Valid results were those in which the internal control gene was amplified with a Ct ≤ 35. A sample was considered positive if at least one of the genes was amplified with a Ct ≤ 40.Viral nucleic acid extractions were made from 200 μL of saliva samples with the QIAamp Viral RNA mini kit  and eluted in 60 μL. Saliva specimens with high viscosity were diluted with an equivalent volume of UTM. Detection of SARS-CoV-2 was performed by RT-PCR with 5 μL of RNA template using the SARS-CoV-2 ELITe MGBThe presence and load of the TTV genome were determined by using a single-step TaqMan in-house real-time PCR, previously developed in our laboratories . ExtractExtracted DNA was amplified with a PCR protocol developed in our laboratories. The amplification was performed by a quantitative Taqman real-time PCR. PCR reaction was performed by Premix Ex Taq™  in a 25 μL volume with 5 μL of extracted DNA and 0.5 μM of each primer . A Taqman probe  was also used at a concentration of 0.1 μM in the PCR reaction. The PCR was carried out using the following program: 95.0 °C for 1 min and then 95.0 °C for 5 s and 60.0 °C for 30 s for a total of 40 cycles. To estimate the reproducibility of the assay, a ReDoV-negative sample spiked with a known copy number of a recombinant plasmid into which the target fragment of real-time PCR was inserted, was repeated in five independent experiments, and the coefficient of variation was calculated. The reproducibility of the PCR assay was good, being the inter-assay variation lower than 0.1 Log and 1.2% in Ct. The lower detection limit was calculated to be of about 10 copies.A segment of 1199-nt within the Rep gene of virus genome  was amplified by a qualitative single-step PCR protocol (ssPCR) by using sense primer  and antisense primer . The PCR was performed on a 25 μL PCR mixture, and the amplified products was electrophoresed on agarose gel  and sequenced with the Big Dye Terminator Cycle Sequencing kit  using only the ReDoV_REP_R2 primer. The sensitivity of ssPCR was previously measured by testing serial dilutions of a positive sample and was about 1000 DNA copies.https://www.megasoftware.net/, accessed on 1 September 2022).The sequences obtained were then visually verified and edited using UGENE software and a fragment of 477 base pairs was selected for phylogenetic analysis. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model and phylogenetic tree were obtained by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model. The tree is drawn to scale, with branch lengths measured in the number of nucleotide substitutions per site. Phylogenetic analyses were carried out in MEGA version 11 software  was used for statistical analysis. Transformed TTV and ReDoV loads in Log format were used for analysis. The Chi-square test and Fisher’s exact test were applied to evaluate the heterogeneity of contingency tables. Differences between distributions were calculated by using non-parametric the Mann–Whitney U test. The association among variables was evaluated by using the analysis of variance test, Tukey’s multiple-comparison test and the Kruskal–Wallis test. Correlations between variables were assessed using the Spearman rho correlation coefficient. All The presence of SARS-CoV-2 RNA was investigated in all 448 saliva samples. All 122 hospitalized subjects with positive SARS-CoV-2 nasopharyngeal swabs also tested positive for SARS-CoV-2 in saliva. All 326 healthy individuals from screening cohort were negative for SARS-CoV-2 both in nasopharyngeal swab (NPS) and saliva samples. The mean SARS-CoV-2 cycle threshold (Ct) value in saliva was 30 .The presence of TTV DNA was investigated in the entire cohort of 448 saliva samples . The ovep = 0.0004, Chi-Square test). A statistically significant difference in ReDoV prevalence was also noted when the saliva samples were grouped by SARS-CoV-2 status: ReDoV was 65% prevalent in the SARS-CoV-2-negative group and 52% in the positive group . When the Ct values for SARS-CoV-2 PCR detection were evaluated, no statistically significant difference was observed relative to ReDoV and/or TTV status (data not shown).Overall, ReDoV DNA was investigated in 377 saliva samples . No significant difference in ReDoV levels was observed in saliva samples with different SARS-CoV-2 status . As showp < 0.016; Kruskal–Wallis test). The mean viral loads of the single infected samples, namely those infected with TTV or ReDoV alone, showed a lower value when compared to dually infected samples, and Tukey’s multiple-comparison test showed that ReDoV single-infected samples resulted in the only true outlier (p = 0.004).Of these 377 samples, 50% (189/377) reacted positively to both TTV and ReDoV PCRs, and 33% (124/377) were positive to only one PCR. Of these latter samples, 67% (83/124) were TTV positive and ReDoV negative, and 33% (41/124) were TTV negative and ReDoV positive. Sixty-four of 377 (17%) samples were negative for both TTV and ReDoV. Then, we stratified the viral loads of the 313 samples according to the number of viruses they harbored, and the Kruskal–Wallis analysis of variance was used. As in r = 0.606; p < 0.0001). Interestingly, 13 saliva samples tested TTV positive , although the corresponding serum samples were below the detection threshold, while the opposite was seen in 7 saliva samples .The levels of TTV and ReDoV were also measured in simultaneous serum samples available from 120 subjects, all negative for SARS-CoV-2 infection. Sixty-nine samples tested TTV positive at copy numbers with a mean ± SD of 4.1 ± 0.6 Log copies/mL of serum . Viral loads in plasma and saliva showed a high positive correlation in individual subjects .


The purpose of this study was to compare early clinical and patient-reported outcomes between robotic assisted (RA) and computer navigation (CN) total knee arthroplasty (TKA).P = .34) or body mass index (P = .09), but a higher proportion of males underwent RA-TKA (P = .03). We recorded hospital knee pain, analgesic usage, length of hospital stay, range of motion, and patient-reported outcome measures postoperatively for both patient cohorts.One hundred and fifty patients were enrolled in this prospective, single-surgeon, cohort study, with 75 patients each receiving CN-TKA or RA-TKA in a consecutive series. There were no differences in patient age (P < .001). RA-TKA patients showed improved range of motion (P < .001) and decreased pain scores (P = .006) on day 1. Subsequent days showed no significant differences. Narcotic usage was lower for the RA-TKA group on day 2 postoperatively (P = .03) and onwards. Total morphine equivalent dose was also significantly lower for the RA-TKA than for the CN-TKA group (P < .001). There was no difference in Forgotten Joint Score (P = .24) or Oxford Knee Score (P = .51) between groups at 2 years postoperatively.Hospital length of stay was shorter for the RA-TKA patients (The use of RA-TKA demonstrated reduced postoperative analgesia usage and length of stay. There were no differences seen between CN-TKA and RA-TKA with respect to clinical outcomes at 2 years after surgery. Total knee arthroplasty (TKA) is a highly successful procedure for patients with end-stage osteoarthritis. However, not every TKA provides a patient with an ideal knee postoperatively . PatientCN systems for TKA were introduced in the 1990s, and adoption of CN has been varied by region over the past 2 decades . BenefitRobotics was first developed for TKA in 1992, but the uptake of this technology was limited by its complexity ,14. In 2The saw blade attached to the robotic arm moves within a preplanned stereotactic boundary, which decreases iatrogenic soft-tissue damage ,19. SimiRA-TKA has been shown to decrease analgesia requirements, length of stay (LOS), and physical therapy requirements immediately after surgery when compared with the use of conventional instrumentation . DecreasTherefore, the purpose of this study was to determine if the choice of surgical instrument (CN or RA) had influence on early clinical and patient-reported outcomes. We hypothesize that there would be a difference between these groups as this is what had been shown between RA-TKA and conventional instruments. We recorded hospital LOS and patient-reported outcome measures (PROMS) at 3 months, 1 year, and at a minimum of 2 years follow-up. Moreover, to understand the relevance of soft-tissue protection aspect of the RA technique, we collected inpatient pain scores and analgesia usage, which may be considered surrogates for soft-tissue trauma.One hundred and fifty patients were enrolled in this prospective, single-center, cohort study. The first group of patients received CN-TKA  between December 2015 and November 2016, while the second group received RA-TKA  between December 2016 and August 2017. All procedures were performed by a single surgeon who has a high-volume TKA practice using both CN- and RA-TKA.Patients were enrolled in this institutional review board-approved study if they were indicated for a unilateral primary total knee replacement, were able to independently complete PROMS, and did not have a history of chronic pain. Patients in each group were sequential cohorts, with the CN-TKA patients being the 75 patients to have TKA by the surgeon immediately before the introduction of RA-TKA. Intraoperatively, all patients received spinal anesthesia with sedation and an adductor canal block with local anesthetic infiltration of joint capsule and skin. Postoperatively, consistent pain management and rehabilitation protocols were applied to all study subjects. All patients received a combination of paracetamol, celecoxib, slow-release oxycodone (5- to 10-mg bd), and fast-release oxycodone (2.5- to 10-mg 2/24 prn). Dosages were titrated to requirements, and alternatives were used if patients did not tolerate the standard regime. There were no changes in postoperative management during the time in which this study was undertaken. Discharge criteria were standardized. Six patients in each group were discharged to a rehabilitation unit, with all others discharged home.Alignment targets for both groups were within a boundary of 5° varus to 3° valgus coronal alignment using an adjusted mechanical axis principle. This was achieved with preresection balancing using the software and virtual balancing abilities of both systems. No bony recuts were performed in either group.The surgeon performed a medial parapatellar approach with minimal soft-tissue releases for exposure. The patella was resurfaced in all cases using a conventional cutting guide. All patients in both groups received cemented tibial and patella components with uncemented femoral components .Demographic information, history, and examination findings were recorded for all cases. In-hospital metrics included blood loss, LOS, range of motion (ROM) in operated leg, patient-reported Visual Analogue Pain Score , and the use of medications. Medications recorded included families of analgesics including opioids, nonsteroidal anti-inflammatory drugs, antiemetic, and anaesthetics. The observers were unaware that their recording in hospital data was being used to assess patient outcomes, and it was recorded as part of routine nursing practice. Opioid dosages given to the patients were converted to oral morphine-equivalent dosages . At 90-dDescriptive statistics were used to describe means and standard deviations for all variables in this study. A sample size calculation was performed using FJS 2 years after surgery as the primary outcome measure. Using existing data from published studies  on TKA, P = .34), BMI , or operative side . There was a higher portion of males who underwent RA-TKA . There was no difference in mean preoperative European Quality of Life in five dimensions score (EQ-5D) , Oxford Knee Score (OKS) , or FJS  between groups. There was no difference in mean preoperative ROM between groups .There was no difference between the CN-TKA and RA-TKA patients in patient age . Opioid requirement was lower for the RA-TKA group on all days of admission. While the difference did not reach statistical significance on day 1 (P = .13), there was a statistically significant decrease in opioid use on days 2-4 (P < .01). Total morphine equivalent use was significantly lower for the RA-TKA than for the CN-TKA group (P < .01)  . There wP < .01). There were no differences seen in blood loss as measured by change in hemoglobin levels.Time needed to complete the surgical procedure was longer for RA-TKA cases  or OKS between groups . There was no significant difference seen in postoperative ROM at either 3 months (P = .912) or 1 year postoperatively (P = .512)  .To our knowledge, this is the first study to compare computer-navigated and robotic TKA. We found less opioid use, reduced pain, and shorter LOS postoperatively in patients who underwent RA-TKA compared with those who underwent CN-TKA. We anticipated finding improved short-term patient outcomes with robotics as robotics has been shown to reduce iatrogenic soft-tissue damage and preserve vulnerable structures in the knee capsule . This adThe diminished opioid requirements with decreased LOS are likely to be beneficial in view of the increased use of day surgery for TKA and the desire to limit narcotic usage . The resThere was an increase in length of procedure between CN-TKA and RA-TKA (16 minutes more with RA-TKA). This would impact operating room efficiency and costs. One of the factors was the increased amount of data collection and research taking part in the RA-TKA cases. Although the learning curve associated with RA surgery has been shown to be short , these tWhile RA-TKA provided benefits in the immediate postoperative period, we did not find a significant difference between CN- and RA-TKA when evaluating postoperative PROMS at any time point between 3 months and 2 years after surgery. Both techniques have been shown to be accurate relative to manual instrumentation and use optic tracking with the ability to verify accuracy intraoperatively ,20,37. GThere are several limitations of this study. The procedures were performed sequentially, with CN-TKA being performed on 75 patients before the commencement of RA-TKA at the institution. The RA-TKA group consists of the surgeon’s first 75 cases using this technique and thus include the learning curve within the group. The patients were not blinded to the technique used. We have not included radiological outcomes to compare RA-TKA patients to CN-TKA patients. This study did not examine overall costs of the 2 techniques, and a health economics comparison including the equivalent longer term outcomes remains warranted. Surgeon preference and ease of use considering both physical and mental fatigue would also be relevant to future comparisons.This study has demonstrated a significant reduction in postoperative opioid requirement and LOS with the use of RA-TKA when compared with CN-TKA. The short-term differences are not maintained, with no difference in patient-reported outcomes or ROM seen between 3 months and 2 years after surgery. Future prospective randomized controlled trials are warranted to prove this.G.W. Clark is in the speakers' bureau of or gave paid presentations for, is a paid consultant for, and receives research support as a principal investigator from Stryker. D. Wood receives royalties from Global Orthopaedics for Absolut hip replacement and receives research support from Zimmer for RCT of onlay vs inset patella.https://doi.org/10.1016/j.artd.2021.11.014.For full disclosure statements refer to


Drug-loaded microspheres are an ideal bone tissue delivery material. In this study, a biodegradable Schiff base chitosan–polylactide was used as the encapsulation material to prepare drug-loaded microspheres as biocompatible carriers for controlled vancomycin release. In this regard, Schiff base chitosan was prepared by the Schiff base method, and then different proportions of the Schiff base chitosan–polylactide polymer were prepared by ring-opening polymerization. Drug-loaded microspheres were prepared by the W/O emulsion method, and the polymers and polymer microspheres were characterized and studied by NMR, IR, and antibacterial methods. The drug loading and release rates of microspheres were determined to investigate the drug loading, encapsulation efficiency, and release rate of drug microspheres at different ratios. In this study, different proportions of Schiff base chitosan–polylactic acid materials are successfully prepared, and vancomycin-loaded microspheres are successfully prepared using them as carriers. This study proves that the materials have antibacterial activities against Staphylococcus aureus and Escherichia coli. The particle size of drug-loaded microspheres was below 10 μm, and the particle size decreased with decreasing molecular weight. The obtained results show that 1:100 microspheres have the highest drug-loading and encapsulation efficiencies, the drug-loaded microspheres have no burst release within 24 h, and the release quantity reaches more than 20%. After 30 days of release, the release amounts of 1:10, 1:20, 1:40, 1:60, and 1:100 drug-loaded microspheres were 64.80 ± 0.29%, 54.43 ± 0.54%, 44.60 ± 0.43%, 42.53 ± 0.40% and 69.73 ± 0.45%, respectively, and the release amount of 1:100 was the highest. Vancomycin is a tricyclic glycopeptide and an antibiotic. Methicillin-resistant Staphylococcus aureus (MRSA) is the most common pathogen in hospital infections. Vancomycin has related antibiotic activity against Gram-positive pathogens, including MRSA . With thTraumatic osteomyelitis or infectious bone defects mostly increase the difficulty of treatment because of the risk of infection in the treatment of multiple loading. Conventional oral or intravenous antibiotic therapy, due to high doses of drugs, easily causes adverse reactions and poor local treatment. The desired antibiotic sustained release carrier material should have a certain drug-loading and stable-release rate. Local administration can enhance drug efficacy while effectively releasing antibiotics to control infection and can effectively avoid adverse effects due to systemic administration ,16. ChitIn this study, different proportions of the Schiff base chitosan–polylactic acid polymer (N-COS-PLA) are prepared from chitooligosaccharides and lactide. Amino Schiff base is generated by amino as the modification site, and the antibacterial activity of chitosan oligosaccharide is improved to the maximum extent. The characterization and performance of N-COS-PLA are studied by NMR, IR, and X-ray diffraction to explore its antibacterial activity against Gram-negative and Gram-positive bacteria. The application value of chitooligosaccharides is improved, and the application field of chitooligosaccharides is expanded. At the same time, chitooligosaccharide hydroxyl groups are connected to polylactic acid to prepare polymer materials, and vancomycin is wrapped to prepare drug-loaded microspheres. The effects of different proportions of polymers on drug loading, encapsulation efficiency, and release amount are studied.Chitooligosaccharides (CAS: 148411-57-8)  were purchased from Shanghai McLin Co., Ltd. . Lactide was provided by Fengyuan Co., Ltd. . All reagents in the experiment were analytically pure and were obtained from Tianjin Damao Co., Ltd. . All chemical reagents were not purified further. Staphylococcus aureus and Escherichia coli were obtained from Luwei Technology Co., Ltd. .The procedure was as follows. Take a certain amount of chitooligosaccharides (COS), dissolved in de-ionized water; add the same amount of methanol solution to induce swelling for 1 h; then, add benzaldehyde and methanol 1:10 mixed solution and continue the reaction at 65 °C for 3 h. After the reaction, cool the solution to room temperature. Using a 5% NaOH solution, adjust the pH to alkaline. Perform the centrifugal collection of solid precipitation. Wash the precipitate with anhydrous ethanol three times. Vacuum dry the precipitate at 45 °C for 24 h. The obtained product is the amino Schiff base chitooligosaccharides (N-COS) .2 was replaced several times; the reaction temperature was 140 °C, and the reaction time was 8 h. After the reaction, the product was cooled to room temperature and then precipitated by adding anhydrous ethanol. The product was obtained by centrifugation. After vacuum drying at 45 °C for 24 h, different proportions of N-COS-PLA were obtained. The reaction synthesis route is shown in N-COS and lactide were added to a small amount of dry DMSO solution at room temperature according to different molar mass ratios . The catalyst stannous octoate with a mass fraction of 0.1% was added. Before addition, NThe vancomycin-loaded microspheres were prepared by the emulsion solvent evaporation method. The appropriate amount of vancomycin was fully dissolved in 5 mL of de-ionized water to prepare the vancomycin solution. The vancomycin solution was dripped into a DCM solution containing composite materials at a high speed of 29,000 rpm/min in a 0 °C ice water bath environment (the water–oil concentration ratio was 1:2). After dripping, the colostrum was formed by continuous high-speed stirring for 5 min, and the colostrum was added to 2% PVA aqueous solution. After dripping, the colostrum was stirred and emulsified at a high speed to form a multiple emulsion. The multiple emulsion was rapidly transferred into an aqueous solution containing 15% isopropanol and stirred at room temperature to completely remove the organic solvent. Solid precipitation was collected and freeze-dried at −50 °C for 24 h to obtain powdered microspheres .The morphology of vancomycin-loaded drug microspheres with different ratios of N-COS-PLA was observed by scanning electron microscopy . The microspheres were numbered according to the different preparation conditions, and the micro-sphere solution was dropped on the clean glass slide and dried naturally. The glass slides were pasted on the carrier table by conductive adhesive, sprayed with gold, and the morphology was observed .1HNMR spectra of the copolymer were determined by NMR . The determination temperature was 25 °C, and the solvent of COS was D2O. The solvent of N-COS was DMSO-d6; the solvent of N-COS-PLA was CDCL3. The molecular weight of the copolymer was determined by gel chromatography  to determine the structure and molecular weight distribution of the copolymer. A comprehensive analysis of the characterization results can determine whether the copolymerization was complete and the residual amount of raw materials [The aterials .−1. The scanning number was 100 times, and the resolution was 4 cm−1. The infrared spectrum of the powder was obtained with a KBr sheet [COS, N-COS, and N-COS-PLA were recorded by a Fourier transform infrared spectrometer . Fourier transform infrared spectroscopy was performed at room temperature in the frequency range of 4000–4500 cmBr sheet .An XRD/max2200 X-ray diffractometer  was operated at a 40 kV voltage and 40 mA current. COS, N-COS, and N-COS-PLA were analyzed at room temperature by scanning 2θ between 10° and 60° in 45 steps for 10 s .2 flow rate: 20 mL/min; heating rate: 10 °C/min [Thermogravimetric curves of COS, N-COS, and N-COS-PLA were determined by a thermogravimetric analyzer . Test conditions: heating temperature range: 50–450 °C; N0 °C/min .COS, N-COS, and N-COS-PLA were dissolved in 10 organic solvents, including water, ethanol, methanol, DCM, EA, petroleum ether, acetone, DMF, DMSO, and acetic acid. A certain amount of material was added to the abovementioned solvent at room temperature, and the dissolution of substances was observed 24 h after ultrasound treatment.The sensitivity of different proportions of polymers to Escherichia coli and Staphylococcus aureus was studied by the filter paper method. First, LB solid medium was configured and cooled to solidification, and 100 μL of bacterial suspension with a certain concentration was evenly coated on the medium surface. The filter paper (10 mm) was soaked in different proportions of polymer solution and sterilized under ultraviolet light. The filter paper was evenly attached to the surface of the culture medium, pre-diffused for 2 h at 4 °C, and cultured in a 37 °C incubator for 24 h. The diameter of the inhibition zone was measured and the average value of each sample was taken three times .The particle size and distribution of microspheres were determined by a laser particle size analyzer . A part of the newly prepared microsphere suspension was diluted to normal light transmission, and the suspension was ultrasonically treated for more than 30 min to make it more evenly dispersed. The suspension was dropped into the sample detection cell of the laser particle-size analyzer, and the particle size and distribution of the microspheres were determined by the particle-size analyzer .The establishment of the standard curve: vancomycin (25 mg) was accurately weighed and dissolved in a 10 mL volumetric flask to prepare a solution for use as a standard solution. The standard solution was diluted to 25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and 300 μg/mL. The absorbance was determined at each concentration . The standard curve was established with the the OD value as the ordinate and the concentration (μg/mL) as the abscissa. The linear range was 25–300 μg/mL.AW and BW are the drug quality in microspheres and the drug dosage.AW and BW are the drug mass in the microspheres and the mass of microspheres.Determination method: A certain amount of microsphere powder was accurately weighed and placed in a centrifuge tube. A small amount of DCM was added to fully dissolve the encapsulation material of the microspheres, and vancomycin embedded in the microspheres was released. Then, a certain volume of PBS was added to dissolve vancomycin, and the encapsulation material N-COS-PLA was precipitated. The solution was centrifuged and a certain volume of supernatant was obtained to determine its OD value. In the experiment, each sample was repeated three times, and the results are expressed as the mean and standard deviation. The measured OD value was taken into the standard curve to calculate the content of vancomycin in the microspheres, and the encapsulation efficiency and drug loading were calculated according to the following formula :EncapsulThe release conditions of microspheres were as follows: 50 mg of microsphere powder was accurately weighed in a 15 mL centrifuge tube, added to 10 mL PBS (pH = 7.4) buffer solution, and continuously released in a 37 °C thermostatic oscillator. At a certain time, the sample was centrifuged, 1 mL of release liquid was extracted from the supernatant, the same amount of fresh release medium PBS buffer solution was added over time, and the constant temperature oscillation was continued. The concentration of vancomycin in a 1 mL release solution was determined by an enzyme-labeled instrument, and the cumulative release of vancomycin in a certain period of time was calculated .The SEM images of N-COS-PLA composite microspheres are presented in 1 HNMR results of COS, N-COS, and N-COS-PLA are presented in 1HNMR spectrum, the peak at 1.98 ppm was attributed to -CH3 in the N-alkylated glucosamine residue, and singlet H peak for N-acetyl glucosamine was observed at 2.98 ppm; there was a multistate peak at 3.45–4.12 ppm, which was due to the chemical shift of -CH- on the sugar ring and proton hydrogen at 6C. The peak at 4.58 ppm was attributed to H of glucosamine [1HNMR analysis of N-COS-PLA, the absorption peaks at 1.67 ppm and 5.27 ppm were the characteristic peaks of CH3 and -CH- in the PLA structure, respectively. The 1HNMR results show that N-COS and N-COS-PLA are successfully synthesized. With an increase in the molecular weight of the polymer, the characteristic peaks -CH3 and -CH- in the structure of PLA in the polymer increased, and the absorption peak intensity of N-COS-PLA at 1.67 ppm and 5.27 ppm increased.The cosamine . In addi−1, which can be attributed to the stretching vibrations of -NH and -OH groups and the hydrogen bonds between and outside COS molecules. The weak band of 2923.83 cm−1 was attributed to the -CH- stretching of COS. The characteristic peaks at 1628.76, 1519.37, and 1322.79 cm−1 were attributed to the carbonyl (C=O) stretching vibration (amide I), amino (N-H) bending vibration (amide II), and amide triple absorption peaks of COS, respectively. The absorption band at 1155.97 cm−1 was produced by C-O-C asymmetric stretching [−1 and 699 cm−1. N-COS-PLA also had strong absorption peaks at 776 cm−1 and 705 cm−1, presenting a sharp peak. The characteristic peak of C=N corresponding to the Schiff base appeared at 1650 cm−1 in FT-IR spectrum, but this peak did not appear in the infrared spectrum of COS. The characteristic peak of the ester group in polylactic acid appeared at 1760 cm−1 of N-COS-PLA. The synthesis of N-COS and covalent binding of N-COS-PLA were confirmed.The infrared characteristic peaks of chitooligosaccharides in the FT-IR spectra  were assretching . The chamax) was 202–298 °C. Polymer N-COS-PLA at 1:10 and 1:20 ratios had the first weight-loss stage; the first weight-loss stage was at 75–135 °C and the mass loss rates were 5.13% and 4.94%, perhaps because the molecular weight of the polymer was lower than that at other ratios, and it was slightly soluble in water; thus, the first stage was the evaporation of water molecules. The other ratios only achieved the second weight-loss stage, which was at 195–405 °C. The mass loss rates were 80.25%, 85.05%, 94.43%, 92.90%, and 95.81%, respectively. The second-stage mass loss rates may have been due to the thermal decomposition of the material. The thermal stability of the material was studied by thermogravimetric analysis. The dissolution of each substance in organic solvents is presented in The inhibition zone diameters of Escherichia coli and Staphylococcus aureus treated with COS, N-COS, and different proportions of N-COS-PLA are presented in Encapsulation efficiency and drug loading are two important criteria for evaluating the drug-loading capacity of microspheres. At the same time, encapsulation efficiency and drug loading are affected by many factors in the preparation of microspheres , such asThe drug-release rate was positively correlated with the molecular weight distribution, morphology, drug loading, and encapsulation efficiency of the material . Figure In the present study, N-COS-PLA polymer materials with different proportions were successfully prepared from chitooligosaccharide and lactide. The introduction of an amino Schiff base can significantly improve the properties of the material, including crystallinity, thermal properties, and solubility. The material had antibacterial effects on Staphylococcus aureus and Escherichia coli, and the molecular weight of PLA had a significant impact on the properties of microspheres. In this study, sustained-release microspheres of vancomycin were prepared by the emulsion crosslinking method. Sustained-release microspheres of vancomycin were prepared by the emulsion crosslinking method. Studies have shown that the particle sizes of drug-loaded microspheres are all below 10 μm, and the particle sizes of microspheres decrease with the decrease in molecular weight. In the experimental study, the drug loading and encapsulation efficiency of 1:100 microspheres were the highest, and there was no burst release within 24 h of drug release. After 30 days of release, the release amounts of 1:10, 1:20, 1:40, 1:60, and 1:100 microspheres were 64.80 ± 0.29%, 54.43 ± 0.54%, 44.60 ± 0.43%, 42.53 ± 0.40%, and 69.73 ± 0.45%, respectively. When the PLA content was the highest, the release amount of 1:100 was the highest. Sustained-release microspheres of vancomycin began to release rapidly in the release medium for 24 h, and the release amount was greater than 20%. The mechanism is that vancomycin hydrochloride molecules contain hydrophilic hydroxyl groups and are water-soluble, which can be combined with water molecules through oxygen bonds and rapidly spread in the solution. In summary, vancomycin sustained-release microspheres showed a good sustained-release effect and antibacterial activity in vitro, which provided a basis for its application in the treatment of bone infection. In the study of vancomycin-loaded materials, the amphiphilic material prepared in this study made the release of vancomycin easier to control and more stable, avoided the side effects of systemic medication, and improved the medication rate.


The intelligent multi-layer film was prepared via layer by layer casting of gelatin, chitosan (added with 0.5% of RCE) and CMC biopolymers. The thickness of the multi-layered film was the highest (0.123 ± 0.001 mm) compared to gelatin-, CMC- and chitosan-based monolayer films (p < 0.05). Chitosan film has the highest tensile strength (p < 0.05), followed by multi-layer, CMC and gelatin films. Elongation at break was slightly higher in CMC (35.67 ± 7.62%) compared to the multi-layer film (33.12 ± 9.88%) and gelatin film (p > 0.05). Water vapor permeability was higher in the multi-layer film (1.244 ± 0.05 × 10−5 g mm h−1cm−2 P−1) than the other monolayer films. Moisture content was highest in chitosan film followed by the multi-layered film (p < 0.05) and then the CMC and gelatin films. CMC film showed the highest solubility compared to multi-layered and chitosan film (p < 0.05). Additionally, transmittance and transparency values in the multi-layered film were the lowest compared to the chitosan-, CMC- and gelatin-based films. L* and a* values were the lowest, while b* values increased in the multi-layered film compared to the other film samples (p < 0.05). pH sensitivity and ammonia gas tests revealed similar color changes in chitosan and multi-layer films. However, FTIR spectra confirmed that dye leaching was not detected for the multi-layered film soaked in ethanol. The biodegradability test showed rapid degradation of multi-layered and chitosan films within 1 month. Based on the optimum results of the multi-layered film, it was applied to monitor the fresh quality of tilapia fish fillets at 4 °C for 10 days. The results of freshness acceptability were noted on day 6 due to the change in color of the multi-layer film with an estimated total volatile basic nitrogen content of 21.23 mg/100 g. Thus, the multi-layered film can be used as an indicator to monitor the quality of the fish freshness without leaching dye onto the food surface.This study aimed to fabricate an intelligent monolayer and multi-layered biodegradable films incorporated with red cabbage extract (RCE) to act as a safe and reliable freshness indicator. A film-forming solution (FFS) of gelatin, carboxymethyl cellulose (CMC) and chitosan was prepared and fortified with 0.5% ( Intelligent packaging has been widely explored to detect the safety and shelf-life of foods susceptible to microbial and chemical spoilage during storage . IntelliPromising biopolymers such as gelatin, chitosan and carboxymethyl cellulose (CMC) have been employed in the preparation of intelligent biodegradable films. Gelatin is a high-molecular-weight biopolymer derived from collagen by thermal degradation . GelatinRoselle calyx, purple eggplant, beetroot, dragon fruit and red cabbage [p-coumaric and caffeic acids [Anthocyanin extracts from plant-based products have been incorporated in pH sensing intelligent films to replace chemically synthesized dyes. Anthocyanins are the natural pigments of vascular plants and are responsible for the pink, red, violet and blue colors of vegetables and fruits such as  cabbage ,11. The ic acids . The staic acids . Anthocyic acids . Gelatinic acids . RCE conic acids . Additioic acids . Due to In this study, we aimed at developing a biopolymer-based, pH-sensitive intelligent film by adding anthocyanin-rich RCE into gelatin-, chitosan- and CMC-based monolayer films and multi-layer (gelatin/chitosan + RCE/CMC) film. The RCE-anchored monolayer and multi-layer films were analyzed for microstructural, physicochemical, mechanical and disintegrable properties. Moreover, the multi-layer film was applied to monitor the freshness of tilapia fish fillets during refrigerated storage.Red cabbage was procured from Makro supermarket in Chiang Rai, Thailand, in May 2022. Commercial bovine gelatin  was obtained from Nutrition SC Co., Ltd. . Dimethyl sulfoxide (DMSO) was purchased from RCI Labscan Limited, Bangkok, Thailand. Chitosan powder , carboxymethyl cellulose (CMC), Tween-80 and plate count agar (PCA) were obtained from Sigma-Aldrich . Glycerol was purchased from Merck . All the chemicals used in this study were of analytical grade.w/v) and were kept overnight at 5 °C. The next day, ethanol was removed using a rotary evaporator, followed by freeze drying  for 48 h, and stored in a light-impermeable plastic bag at −20 °C until use for incorporation in intelligent film fabrication.Red cabbage samples without damage were brought to the Food Technology Laboratory at Mae Fah Luang University, Chiang Rai, Thailand, and washed with distilled water to remove dust and dirt. Red cabbage extract (RCE) was prepared according to Pereira et al.  with sliw/v) of RCE. Briefly, gelatin , CMC  and chitosan  were added with glycerol 0.5% (w/v) in 100 mL for each biopolymer film. All the film-forming solutions (FFS) added with RCE 0.5% (w/v) were thermally dissolved, followed by pouring into molds and drying under fan for 24 h. Similarly, three layers were cast step-wise to develop a multi-layer film using gelatin, CMC and chitosan biopolymers. A porous gelatin-based layer as the innermost layer was prepared by using the micro bubble technique [w/v) was mixed with 100 mL of distilled water and 0.5% (w/v) glycerol. The mixture of gelatin FFS for the inner layer was dissolved at 80 °C for 15 min. The mixture was cooled before homogenization to generate bubbles at 20,000 rpm for 3 min. Gelatin FFS was then poured into the mold and kept overnight at 4 °C. The chitosan FFS for the middle or pH-sensitive layer incorporated with RCE was prepared by dissolving 0.5% (w/v) glycerol and 2% (w/v) chitosan in 100 mL of distilled water, and stirring was continued overnight and then added with 0.5% (w/v) of RCE before pouring it into the mold over the porous gelatin layer. Finally, it was left under a fan for 24 h before pouring the outer layer. The CMC-based FFS for the non-porous outer layer was produced by mixing 100 mL of distilled water with 0.5% (w/v) glycerol and 2% (w/v) CMC and dissolved at 60 °C for 20 min. The CMC-based FFS of the non-porous outer layer was cast on pre-casted layers of the same mold and allowed to dry under a fan for 24 h. All the monolayer and multi-layer film samples were finally conditioned in a humidifier at relative humidity (RH) of 45% with a humidifier temperature of 25 °C [Gelatin-, CMC- and chitosan-based monolayer films were developed and added with 0.5%  at nine random positions on the film as described by Muhammad et al. . The mic2 and conditioned at 50% RH at 25 °C for 48 h. The test was carried out using a cross-head speed of 30 mm/min, a 1 kN load cell and an initial grip length of 30 mm .The tensile strength (TS) and elongation at break (EAB) of films were determined by a Universal Testing Machine . Before testing, each film sample was cut into sizes of 20 × 50 mmThe WVP of the films was measured by using a modified ASTM method (1989) as described by Kaewprachu et al. . All the2); t is the time of film exposure (h); (P2 − P1)−1 is the vapor pressure differential across the film (Pa). The WVP is expressed as g mm h−1 cm−2 Pa−1.W is the weight gain of the cup (g); X is the film thickness (mm); A is the area of exposed film  for 24 h. The un-dissolved debris were then removed by centrifugation at 3000× g for 30 min. The residual film samples were dried in a hot air oven set at 105 °C for 24 h and weighed to determine the solubility of the film by Equation (2):i is the initial weight of the film and Wf is the final weight of the film after drying.The film solubility was determined according to the method of Kaewprachu et al.  with sli600 = absorbance at 600 nm; X = average thickness of the film.The color values of the film were determined using Color Quest XE . The values of L* (lightness), a* (red-green) and b* (yellow-blue) were recorded. The UV light barrier properties of the films were measured  using a 2 film was placed in a small Petri dish to be submerged in pH buffer solution (pH 1 to 12) prepared using 0.1 M and 0.2 M hydrochloric acid, 0.1 M and 0.2 M sodium hydroxide, 0.2 M potassium chloride, 0.1 M acetic acid, 0.1 M sodium acetate, and 0.1 M disodium hydrogen phosphate. After 10 min, photographs of the film samples were recorded. NH3 was used to analyze gas sensitivity based on the color response of the chitosan and multi-layer film samples to basic gas. Briefly, the films (10 × 10 mm2) were placed in an Erlenmeyer flask (500 mL) containing ammonia solution for 30 min at 25 °C. The photos of the films were captured using an optical scanner  and the stability of the films was determined with the color change [The pH sensitivity of the film was determined according to the method described by Pereira Jr, de Arruda and Stefani  with somr change .2 film samples in glass jars containing soil compost. Film samples were incubated under a controlled temperature and relative humidity of 58 ± 2 °C and 50 ± 10% RH [Dye leaching from the film samples was evaluated as per the method of Lee et al. . Accordi± 10% RH  for a pe2) was fixed on the headspace of sterile Petri plates containing 25 g of fish fillet samples. The plates were then closed tightly with paraffin film and placed in an incubator at 4 °C for a period of 10 days. Chemical and microbial analyses were every two days of storage.To perform the application test on fish, the developed multi-layered film  was analyzed to indicate the microbial quality and freshness of fish samples . Every 2Statistical analysis was performed using SPSS for Windows  to test the analysis of variance (ANOVA). At a 95% confidence level, Duncan’s multiple range tests were used to identify the significant difference between treatments. Statistical analysis was performed for physical properties  and mechanical properties ; the solubility of the films was also analyzed statistically as a study variable.p > 0.05) (w/v) of RCE showed the highest thickness of 0.123 mm compared to the monolayer films (p < 0.05). Film thickness could improve the light barrier, the water vapor transmission rate and mechanical properties. Chitosan film supplemented with 0.3% of black soybean seed coat extract exhibited a higher thickness than plain chitosan film [The thickness of the monolayer films prepared from gelatin, CMC and chitosan ranged from 0.034 mm to 0.040 mm ( > 0.05) . The mulsan film . Polyphesan film . Thus, tThe microstructure of the film surface morphology observed under a scanning electron microscope presented smooth surfaces for CMC, chitosan and multi-layer films, indicating the high compatibility of molecular interactions between the materials . Howeverw/w) of Chinese bayberry (Myrica rubra Sieb. et Zucc.) into the cassava film matrix resulted in a dense and compact internal microstructure, which greatly improved the water vapor permeability and tensile strength of the film [As for the mechanical properties of the film, the highest tensile strength of the monolayer chitosan incorporated with anthocyanin was 21.76 MPa . Accordithe film .Regarding the water vapor permeability (WVP) of the film, it was noticed that the multi-layered film showed the highest response when compared to the monolayer film samples . AccordiCaesalpinia sappan L.) heartwood extract incorporated in gelatin film [Brassica oleraceae) as time–temperature indicators for application in intelligent food packaging [The lowest transmittance and transparency values were obtained in multi-layer film, indicating the partial opacity of three distinct layers, as shown in tin film . The decackaging . Kaewpraackaging  reportedRCE powder was tested for pH sensitivity, as shown in Gas sensitivity tests of the chitosan film and multi-layer film were analyzed as shown in −1 and 3267.01 cm−1, indicating the stretching vibration between N-H and O-H bonds due to the interaction between the chitosan and the anthocyanin. When the same chitosan film was soaked in the ethanol solution, shifts in the chitosan peaks were observed at values from 3291.31 cm−1 and 1027.05 cm−1 to 3330.98 cm−1 and 1053 cm−1, respectively. The presence of the peaks at 3418 cm−1, 1639 cm−1 and 1053 cm−1 showed the possibility of the presence of the functional groups of anthocyanins [Caesalpinia sappan L.) heartwood extract can form a hydrogen bond with a related functional group to reduce free hydrogen that can form hydrophilic bonds with water [Dye leaching is another important parameter that was analyzed in monolayer and multi-layered intelligent films. According to the Food and Drug Administration, the most common food stimulants for fresh and refrigerated fish are water and ethanol. Chitosan and multi-layer film samples were soaked in the respective aqueous and ethanol solvents for 10 days. The leaching of RCE dye normally occurs in monolayer film samples. So, this test was carried out to verify and assure the safe use of the multi-layer intelligent film for food applications. Then, the FTIR analysis for both the dried films and the solution samples was performed. FTIR spectra were analyzed to verify or spot the presence of the functional group of the anthocyanins in both the dried film and the solution sample in which the film was soaked for 10 days. The FTIR analysis for the film was to ensure that anthocyanins from RCE were retained in both the monolayer and the multi-layer film. Similarly, FTIR analysis was performed for the residual solution to verify the leached anthocyanins into the solution when exposed to different food stimulants. ocyanins . Compari−1. However, after the sample solution was analyzed, new peaks were observed at 3266.94 cm−1 and 1634.70 cm−1, indicating shifts caused in the peaks due to the interactions between the anthocyanins and the polymer matrices, thereby indicating that the dye was leached from the multi-layer film as well when water was used as a stimulant. Moreover, when the multi-layered film was soaked in ethanol and water, the film solution of ethanol gave a negative result, indicating that no dye was leached out despite the multi-layer films being soaked in ethanol solution for 10 days. As shown in −1, 1044.02 cm−1 and 1380.26 cm−1. When the solution sample was analyzed, no specific peaks were obtained and the graph showed a stable line, indicating the absence of anthocyanins being leached from the multi-layer film. pH-sensitive polymeric dyes were fabricated by grafting covalently phenol red and rosolic acid onto the chitosan matrix and showed excellent color stability and dye leaching resistance [Similarly, the multi-layer film was also soaked in both water and ethanol as stimulants. Using water as the solvent, even the multi-layer film showed positive test results. As can be seen from sistance .Biodegradability was confirmed by the disintegration of gelatin, CMC, chitosan monolayer and multi-layer intelligent films visualized under an optical microscope, as shown in The freshness and microbial quality of Nile tilapia fillets were monitored by using intelligent film samples incorporated with RCE and stored at 4 °C for 10 days A. The coThe production of basic volatile nitrogen in the form of ammonia gas has been reported in degraded silver carp fish muscles during storage . The mul3), collectively known as total volatile basic nitrogen, were detected using plastic film incorporated with bromophenol dye as a pH-sensing indicator of fish freshness, placed in the head space of the package, showing a gradual color change and increased bacterial load during fish spoilage [TVB-N value is widely used as a quality parameter to evaluate the freshness of fish and meat products . The avespoilage .We developed biopolymer-based intelligent monolayer and multi-layered films incorporated with RCE to act as a natural pH-sensing indicator for food applications. Gelatin-, chitosan- and CMC-based monolayer films and a multi-layered intelligent film were tested for their physico-chemical, microstructural and mechanical properties. The multi-layered film yielded the highest results compared to gelatin-, CMC- and chitosan-based monolayer films. The microstructure analysis revealed thicker cross-sections in the multi-layer film than the other films. Chitosan film has higher tensile strength than the multi-layer film, CMC and gelatin films. Elongation at break was slightly higher in CMC compared to the multi-layer film and gelatin film. WVP was higher in the multi-layer film than the other monolayer films. Moisture content was highest in chitosan film, followed by the multi-layered film and other monolayer films. CMC film showed the highest solubility compared to multi-layered and chitosan films. Additionally, transmittance and transparency values in the multi-layered film were the lowest compared to the chitosan-, CMC- and gelatin-based films. L* and a* values decreased, while b* values increased in the multi-layered film compared to the other film samples. pH sensitivity and ammonia gas tests showed no changes in the chitosan and multi-layer films. FTIR spectra confirmed that dye leaching was not detected in the ethanol-soaked multi-layered film. Biodegradability tests showed rapid degradation of multi-layered and chitosan films within 1 month. Therefore, the multi-layered film was employed to monitor the fresh quality of tilapia fish fillets at 4 °C for 10 days. The results of freshness acceptability were noted on day 6 due to a change in the color of the multi-layered film and as evidenced by higher TVB-N content of 21.23 mg/100 g in stored tilapia fish fillets. Thus, the multi-layered film could be used as a natural and safe indicator to sense the quality of the fish freshness without leaching dye onto the fish muscle.